The invention relates to a solution used in analysis of biological tissues and, more particularly, to the composition of an aqueous clearing solution making biological tissues transparent.
For some lift sciences such as cell biology, neurobiology, molecular biology, physiology, immunology, signal biology, and development biology, to examine biological or non-biological planar or three-dimensional microscopic structures, in addition to having a good microscope such as a confocal microscope to obtain high-resolution images, it is also necessary to specially take care of preparation of samples, recording of microscopic images, and processing of images so that optics can be exploited to examine some samples and biological cells or tissues capable of emitting fluorescence. When preparing samples, it is necessary to assure the wholeness of three-dimensional structures of the samples in the processing procedure such as the fixation or mounting procedure.
Generally speaking, conventional transmitted light microscopy is extensively used in almost every biological laboratory for observation of cellular structures. Biological tissues are usually stained with dyes before they can be examined with microscopy. To view the stained internal structures, tissues are usually sectioned into thin slices. In order to increase tissue transparency, most preparations of stained samples are cleared by dehydration and lipid extraction processes. For example, xylene is often used to clear paraffin sections after alcohol dehydration. As a result, the stained internal features can be sharply focused.
However, solvent extraction requires more processing time and often causes morphology distortion and needs to be avoided in some situations. For example, cryosectionings performed for rapid diagnosis purpose are usually not fixed well. These tissue slices are usually embedded directly in glycerol-based aqueous mountant without clearing process to avoid destruction of the fragile morphology. The resulting microscopic images are thus suffered from decrease of clarity. Moreover, during the processes of examination, scanning, and image reconstruction of biological tissues, the reconstructed thickness can only reach 100xcx9c200 micrometers due to not good transparency of tissue samples.
Accordingly, the present invention proposes an aqueous clearing solution to effectively make biological tissues transparent without damaging tissues and without dehydration process. FIGS. 1A to 1C demonstrate that an insect brain of about 500 xcexcm thick that is normally opaque in saline (FIG. 1A), semi-opaque in 80% glycerol-saline mixture (FIG. 1B) and completely transparent in the invented clearing solution (FIG. 1C).
The primary object of the present invention is to provide an aqueous tissue clearing solution making biological tissues transparent without dehydration process.
Another object of the present invention is to provide a method to process biological tissue processed by an aqueous tissue clearing solution to obtain images of higher resolution in fluorescent and non-fluorescent light microscopes.
Yet another object of the present invention is to provide an aqueous tissue clearing solution to make tissues transparent without damaging slices and detailed morphology of tissues.
To achieve the above objects, an aqueous tissue clearing solution of the present invention comprises one or more of dimethyl sulfoxide, diatrizoate acid, ethylenediaminetetraacetic acid, glucamine, xcex2-nicotinamide adenine dinucleotide phosphate, sodium diatrizoate, and derivative of polyoxyalkalene (with a trade name of Tween 20 and used as emulsifying agent and detergent) in a suitable aqueous solution. This aqueous tissue clearing solution is utilized to make tissues transparent.